Functional/Structural Studies of Mammalian Neuraminidase

描述（由申请人提供）： 该提案的范围是研究哺乳动物溶酶体神经氨酸酶（NEU 1）在正常细胞代谢中的功能，以及其在人类疾病中的损失的后果。NEU 1属于普遍存在的唾液酸酶超家族。哺乳动物神经氨酸酶包括胞质、溶酶体和质膜异构体，这些水解酶的生理作用的线索，特别是NEU 1，最近才出现。NEU 1通过去除末端唾液酸残基来启动唾液酸-糖缀合物的水解。该酶在唾液酸酶中是独特的，因为它必须与保护性蛋白/组织蛋白酶A（PPCA）结合用于细胞内路由和溶酶体活化。最后，NEU 1与2种神经退行性代谢疾病有关：唾液酸沉积症由NEU 1的结构性病变引起，半乳糖唾液酸沉积症（GS），NEU 1和β-半乳糖苷酶的联合缺乏，由PPCA的缺乏引起。这些研究基于三个具体目标。目的1：研究NEU 1和PPCA之间的结构-功能关系. PPCA前体的3D结构将用于靶向2种蛋白质之间的潜在接触位点的诱变以及在唾液酸沉积症患者中鉴定的NEU 1突变。我们还将使用跨越全长PPCA和NEU 1的重叠肽来鉴定NEU 1/PPCA相互作用、细胞内转运和激活的关键结构域。这些生化研究将被耦合，以确定Neu 1/PPCA复合物的三维结构。在目标2中，我们将比较Neu 1 ~*~小鼠和PPCA^~小鼠的特征，以确定唾液酸沉积症和GS的分子基础，并确定Neu 1在正常细胞生理学中未知的功能。在目标3中，我们将在两种模型中实施各种酶替代治疗方法，以评估这些疾病中全身表型的纠正。我们有能力发展这一研究方向，因为我们已经为拟议的研究建立了适当的遗传和生化系统，并且可以依靠杰出的结构生物学家的专业知识来进行项目的晶体学部分。我们的总体目标是更广泛地了解NEU 1在正常生理学和神经退行性疾病唾液酸中毒和GS的病理生理学中的功能。这些疾病主要影响婴儿和儿童。这些研究的结果应该增加我们对NEU 1功能的了解，并改善未来治疗这些灾难性疾病的儿科患者的设计。

DESCRIPTION (provided by applicant): The scope of this proposal is to study the function of mammalian lysosomal neuraminidase (NEU1) in normal cell metabolism, and the consequences of its loss in human diseases. NEU1 belongs to the ubiquitous superfamily of sialidases. Mammalian neuraminidases include cytosolic, lysosomal, and plasma membrane isoforms, and clues about the physiologic roles of these hydrolases, in particular NEU1, have emerged only recently. NEU1 initiates the hydrolysis of sialo-glyconjugates by removing terminal sialic acid residues. The enzyme is unique among sialidases in that it must be associated with protective protein/cathepsin A (PPCA) for intracellular routing and lysosomal activation. Finally, NEU1 is linked to 2 neurodegenerative diseases of metabolism: Sialidosis is caused by structural lesions in NEU1, and galactosialidosis (GS), a combined deficiency of NEU1 and (3-galactosidase, is caused by the absence of PPCA. The proposed studies are based on 3 Specific Aims. In Aim 1, we will investigate the structure- function relationship between NEU1 and PPCA. The 3D structure of the PPCA precursor will be used to target mutagenesis of potential contact sites between the 2 proteins and NEU1 mutations identified in patients with sialidosis. We will also use overlapping peptides that span the full-length PPCA and NEU1 to identify domains crucial for NEU1/PPCA interaction, intracellular transport, and activation. These biochemical studies will be coupled to determine the 3D structure of the Neu1/PPCA complex. In Aim 2, we will compare the characteristics of Neu1~*~ mice and PPCA^~ mice to determine the molecular bases of sialidosis and GS and to identify yet unknown Neu1 functions in normal cell physiology. In Aim 3, we will implement various enzyme replacement therapy approaches in both models to assess the correction of the systemic phenotypes in these diseases. We are in position to develop this line of investigation, because we have established appropriate genetic and biochemical systems for the proposed studies and can rely on the expertise of an outstanding structural biologist for the crystallography part of the project. Our overall goal is to gain a broader understanding of NEU1 function in normal physiology and in the pathophysiology of the neurodegenerative diseases sialidosis and GS. These diseases affect primarily infants and children. Findings from these proposed studies should increase our knowledge about NEU1 function and improve the design of future therapies for pediatric patients with these catastrophic diseases.